Methods of identifying agents that affect cleavage of amyloid-beta precursor protein

ABSTRACT

The present invention provides methods of identifying agents that affect the cleavage of amyloid-β precursor protein (APP) and related vectors, cells and kits, as well as agents identified by the method.

BACKGROUND OF THE INVENTION

[0001] Alzheimer's disease is a degenerative brain disorder that ischaracterized clinically by progressive loss of memory and cognitiveimpairment. Pathologically, the disease is characterized by lesionscomprising neurofibrillary tangles, cerebrovascular amyloid deposits,and neuritic plaques. The cerebrovascular amyloid deposits and neuriticplaques contain amyloid-β peptide. The aggregation of amyloid-β peptideis instrumental in the pathogenesis of Alzheimer's disease.

[0002] Amyloid-β peptide is derived from amyloid-β precursor protein(APP). APP is a cell-surface protein with a large N-terminalextracellular sequence, a single transmembrane region (TMR) and a shortC-terminal cytoplasmic tail. APP is processed by proteolysis in allcells. Initially, α- and β-secretases cleave APP at definedextracellular sequences just outside of the TMR to release a largeN-terminal extracellular fragment. Thereafter, γ-secretase cuts APP inthe middle of the TMR to generate small extracellular peptides and aC-terminal fragment comprising half of the TMR and the full cytoplasmictail. See, e.g., Selkoe (1998) Trends Cell Biol. 8, 447-453; Bayer etal. (1999) Mol. Psychiatry 4, 524; Haass et al. (1999) Science 286,916-919; Price et al. (1998) Ann. Rev. Genet 32, 461-493

[0003] Cleavage of APP by β- and γ-secretase produces the amyloid-βpeptides (AP40 and AP42) implicated in the pathogeneses of Alzheimer'sdisease. Various methods for the diagnosis and monitoring of the diseaseinvolve assessing the cleavage of APP and detection of amyloid-βpeptide. However, these methods suffer from various disadvantagesincluding the insolubility of amyloid-β peptide, cross-reactivity ofantibodies against the peptide with the precursor APP, and differentlevels of protease activity in different body fluids.

[0004] The γ-cleavage of APP is mediated by presenilins, intrinsicmembrane proteins that may correspond to γ-secretase and that aremutated in some cases of familial Alzheimer's disease. See, e.g., Esleret al. (2000) Nat. Cell. Biol. 2, 428-434. Also, γ-cleavage occurs inAPP-homologs that are not implicated in Alzheimer's disease. Forexample, Notch proteins are membrane proteins that are also cleaved inthe middle of the TMR in a presenilin-dependent reaction. See, e.g. Yeet al. (1999) Nature 398, 525-529; De Strooper et al. (1999) Nature 398,518-522; Struhl et al. (1999) Nature 398, 522-525. Notch proteins arecell-surface proteins involved in intercellular signaling in whichpresenilin-dependent cleavage liberates a cytoplasmic fragment thatfunctions in nuclear transcription. Struhl et al. (2000) Mol. Cell 6,625-636. Sterol regulatory element binding proteins (SREPPs) are alsocleaved in the TMRs to generate nuclear transcription factors. Brown etal. (2000) Cell 100, 391-398. In contrast, the physiologicalsignificance of γ-cleavage of APP, and in particular the biological roleof the cytoplasmic tail fragment, has heretofore been unclear. Inaccordance with the present invention it has been discovered that thecytoplasmic tail forms a functional complex with nuclear proteins, andthat the complex is a potent stimulator of transcription. The presentinvention thus provides new methods for identifying agents that affectthe cleavage of APP.

SUMMARY OF THE INVENTION

[0005] The present invention provides a method of identifying an agentthat affects the cleavage of APP comprising contacting a cell containingAPP modified in the C-terminal cytoplasmic tail to allow detection ofnuclear localization with a candidate agent and measuring nuclearlocalization of a C-terminal cytoplasmic cleavage product of APP in thepresence and absence of the agent.

[0006] In another embodiment, the present invention provides a method ofidentifying an agent that affects the cleavage of APP comprisingproviding a cell containing APP and a protein that interacts with theC-terminal cytoplasmic cleavage product of APP to regulatetranscription, wherein the protein is modified to allow detection ofnuclear localization of the C-terminal cytoplasmic cleavage product ofAPP; contacting the cell with a candidate agent; and measuring nuclearlocalization of the C-terminal cytoplasmic cleavage product of APP inthe presence and absence of the agent.

[0007] The invention further provides agents identified by the foregoingmethod, and compositions comprising the agents.

[0008] In another embodiment, the invention is directed to vectors,transfected cells and kits useful for identifying an agent that affectsthe cleavage of APP.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1A-C show results of Gal4-transactivation assays in PC12cells (FIG. 1A) HEK293 cells (FIG. 1B) and the domain structures ofproteins encoded by the plasmids (FIG. 1C). Test plasmids are identifiedby numbers below the bars in FIGS. 1A and 1B. Luciferase activity wasnormalized for β-galactosidase activity to control for translationefficiency. In FIG. 1A, luciferase activity was additionally normalizedfor the activity of cells co-transfected with Gal4 alone. FIGS. 1A and1B show results from representative experiments.

[0010] FIGS. 2A-D show results of Gal4-transactivation assays (FIGS.2A-C) obtained with the constructs depicted (FIG. 2D). All bar diagramsexhibit representative experiments in the cell types identified to theright of the panel.

[0011]FIG. 3 depicts an immunoblot of COS cells transfected withAPP-Gal4 fusion proteins. The positions of full-length APP-Gal4, theα/β- and γ-cleavage products of APP-Gal4 (APP α/β-Gal4 and APP γ-Gal4,respectively) and the cytoplasmic tail of APP-Gal4 (APPct-Gal4) areindicated on the right. Numbers on the left show positions of molecularweight markers.

[0012] FIGS. 4A-C show transactivation of transcription measured withGal4-fusion proteins and a Gal4-dependent reporter plasmid (FIG. 4A) ascompared with transactivation obtained with LexA-fusion proteins and aLexA-dependent reporter plasmid (FIG. 4B). The structures of theproteins co-expressed with the reporter plasmids and the controlβ-galactosidase vector are shown in FIG. 4C. Transcriptional activationis expressed as fold increase over transcription obtained with theDNA-binding proteins expressed alone.

[0013] FIGS. 5A-E show transactivation using full length APP-Gal4 (FIG.5A) and APP-LexA (FIG. 5B) fusion proteins co-transfected withcorresponding reporter plasmids and a β-galactosidase control plasmids.Various Fe65 proteins described below the bar diagrams wereco-transfected with the test and control plasmids. Transactivation isexpressed as fold induction over transfection with Gal4-APP or LexA-APPalone. The domain structure of Fe65 and deletion mutants of Fe65 areshown in FIG. 5E. FIGS. 5C and D show results of immunoprecipitationscarried out with antibodies to the myc-epitope (FIG. 5C) or to APP (FIG.5D; PIS=preimmune serum) and analyzed by immunoblotting with antibodiesto APP (FIG. 5C and lower part of FIG. 5D) or to myc (upper part of FIG.5D). The positions of the various proteins are indicated on the right ofthe immunoblots, and the locations of molecular weight standards areshown on the left.

[0014] FIGS. 6A-F demonstrate the interaction of Fe65 with Tip60. FIG.6A shows results of a quantitative yeast two-hybrid assay. FIG. 6B showsGST-pulldowns of myc-tagged Fe65 expressed in COS cells with wild-typeand mutant GST-Tip60 proteins. FIGS. 6C-F show immunofluorescencelocalization of tagged HA-Fe65 and myc-tagged Tip60 co-transfected intoHeLa cells. FIG. 6C shows an overview of two adjacent cells; FIGS. 6D-Fdisplay individual and merged labeling patterns. Calibration bars=10 μM.

[0015]FIG. 7A shows the results of transactivation of Gal4-Tip60 in COScells co-transfected with full length or mutant Fe65 proteins and/orwild-type or mutant APP proteins. FIG. 7B shows the domain structure ofGal4-Tip60.

[0016]FIG. 8 provides a model for nuclear signaling mediated by theγ-cleavage product of APP in which APP is cleaved by α-, β- andγ-secretases, releasing the 47 residue cytoplasmic tail plus 10-12hydrophobic residues from the TMR. The cytoplasmic tail complexed withFe65 is translocated to the nucleus and interacts with Tip60 to regulatetranscription.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Proteolytic processing of APP produces, inter alia, amyloid-βpeptide that contributes to the pathogenesis of Alzheimer's disease, anda C-terminal cytoplasmic tail of APP of unknown function until thepresent invention. In accordance with the present invention, it has beendiscovered that the C-terminal cytoplasmic tail of APP engages innuclear signaling. In particular, it has been discovered that thereleased cytoplasmic tail of APP forms a functional complex with thenuclear protein Fe65 and the histone acetyl-transferase Tip 60, and thatthe complex is a potent stimulator of transcription by heterologousDNA-binding domains. This understanding of the role of the cytoplasmictail of APP in nuclear signaling has led to the development of an assayto identify agents that affect the cleavage of APP. Such agents areuseful as candidate therapeutics for the treatment of Alzheimer'sdisease, and as models for rational drug design.

[0018] In one embodiment, the present invention provides a method ofidentifying an agent that affects the cleavage of APP comprisingcontacting a cell, wherein the cell contains APP modified in theC-terminal cytoplasmic tail to allow detection of nuclear localization,with a candidate agent and measuring nuclear localization of theC-terminal cytoplasmic cleavage product of APP in the presence andabsence of a candidate agent. An agent that increases or decreases thenuclear localization of the cleavage product relative to nuclearlocalization in the absence of the agent is defined as an agent thataffects cleavage of APP.

[0019] The term APP as used herein includes naturally occurringmammalian APP and also APP that has been modified, for example in a wayto facilitate measurement of nuclear localization of a cleavage product.Naturally occurring human APP is a 695 amino acid protein, in which theC-terminal 47 residues are designated the cytoplasmic tail. The geneencoding APP, its splice variants, and resulting nucleotide and aminoacid sequences are known in the art and disclosed for example by Kang etal. (1987) Nature 325, 733-736, Selkoe, supra; Bayer et al., supra;Haass et al., supra; and Price et al., supra, the disclosures of whichare incorporated herein by reference.

[0020] Further, APP as defined herein may include other modificationssuch as insertions, deletions and substitutions provided that thefunctions of ability of the cytoplasmic tail or part thereof to becleaved from the remainder of APP and translocated to the nucleus areretained.

[0021] The C-terminal cytoplasmic tail of APP is modified allowdetection of nuclear localization. The modification may be in any regionof the cytoplasmic tail. The modification may be at the C-terminal orN-terminal end of the tail, for example at the junction of thetransmembrane and cytoplasmic domains. In one embodiment, thecytoplasmic tail of APP is modified to include the DNA binding domainand the activation domain of the same or different heterologoustranscription factors. Heterologous as used herein means not derivedfrom a gene encoding APP. In this embodiment, nuclear localization ismeasured by determining activation of transcription of an indicator genethat is under the transcriptional control of a binding site for the DNAbinding domain. Transcription factors and their component DNA-bindingand activation domains are well-known in the art.

[0022] In a preferred embodiment, the cytoplasmic tail is modified toinclude a heterologous DNA-binding domain such as the DNA-binding domainof the yeast transcription factor Gal4, or the bacterial LexA DNAbinding domain. The Gal4 and LexA DNA binding domains are known in theart and disclosed for example by Giniger et al. (1985) Cell 40, 767-774and Hurstel et al. (1986) EMBO J. 5, 793-798. The modification mayfurther contain the transcriptional activation domain of Gal4, oranother activator such as the viral VP16 activator, which is disclosedfor example by Stringer et al. (1990) Nature 345, 783-786. In apreferred embodiment, the cytoplasmic tail of APP is modified to includeGal4 and VP16. A transcription factor module of Gal4-VP16 is describedby Sadowski et al. (1988) Nature 335, 563-564. In an alternativeembodiment, a DNA-binding domain but not an activation domain isincluded with the cytoplasmic tail and the mammalian nuclear multidomainprotein, Fe65, is added as a co-factor to facilitate transcriptionalactivation. Fe65 is known in the art and disclosed for example by Duilioet el. (1991) Nucleic Acids Res. 19, 5269-5274. The term Fe65 as usedherein includes modifications such as insertions, deletions andsubstitutions provided that the function of the ability of Fe65 tofacilitate transcriptional activation is maintained. For example, it hasbeen demonstrated herein that the N-terminal third of Fe65 may bedeleted without loss of this function.

[0023] Accordingly, the modification of the cytoplasmic tail may consistof a heterologous DNA-binding domain, or a module consisting of aDNA-binding domain and a transcriptional activation domain, which may befrom the same or different sources.

[0024] The indicator gene is operably linked to a binding site for theDNA-binding protein. For example, the indicator gene may be provided inthe form of a Gal4 or LexA dependent reporter plasmid containing anindicator gene such as luciferase or chloramphenicol acetyl transferaseunder the control of a Gal4 or LexA regulatory element, respectively,such as an upstream activating sequence. Translocation of thecytoplasmic tail of APP to the nucleus results in translocation of thetranscription factor as well, resulting in activation of transcriptionof the marker gene. Accordingly, detection of the marker gene productprovides an assay for nuclear localization of the cytoplasmic tail ofAPP, and hence measures cleavage of APP. Transcriptional activationassays are described by Fields et al. (1989) Nature 340, 245-246, thedisclosure of which is incorporated herein by reference. Gal4 and LexAreporter plasmids are described by Lillie et al. (1989) Nature (London)338, 38-44 and Hollenberg et al. (1995) Mol. Cell. Biol. 15:3813-3822.

[0025] Candidate agents that may be tested by the assays of the presentinvention include proteins, peptides, non-peptide small molecules, andany other source of therapeutic candidate agents. The agents may benaturally occurring or synthetic, and may be a single substance or amixture. Screening may be performed in high throughput format usingcombinatorial libraries, expression libraries and the like. Agentsidentified as affecting APP cleavage may be subsequently tested forbiological activity and used as therapeutics or as models for rationaldrug design.

[0026] Cells useful for the assays of the present invention includeeukaryotic cells in which the cytoplasmic tail cleavage product of APPcan be translocated to the nucleus. Suitable cells include, for example,insect and mammalian cells. Preferred cells include Schneider, PC12,COS, HeLa and HEK293 cells.

[0027] Cells containing APP may be cells stably or transientlytransfected with a construct encoding APP as described above usingmethods known to those of ordinary skill in the art. Constructscontaining chimeric genes comprising a promoter operably linked tonucleic acid encoding APP and modified to include the DNA-binding domainof a transcriptional activator, or a module comprising the DNA-bindingdomains and transcriptional activation domain of the same or differenttranscription factor, are constructed using well-known recombinant DNAmethods. These constructs are co-transfected into cells with thecorresponding reporter constructs described above. For cases in whichthe construct does not contain a transcriptional activation domain,cells are also co-transfected with vector comprising a nucleic acidencoding Fe65 operably linked to a promoter. The promoter may beconstitutive or inducible.

[0028] The transfected cells are contacted with an agent to be testedfor its ability to affect APP cleavage. A detectable increase ordecrease in nuclear localization of the C-terminal cytoplasmic tail, asmeasured by a change in transcriptional activation of the indicatorgene, is indicative of an agent that affects cleavage of APP. The cellsmay be contacted with the candidate agent before expression of modifiedAPP is induced from an inducible promoter.

[0029] In a particularly preferred embodiment of the present inventionhuman APP is modified to include Gal4-VP16 within the cytoplasmic tail.In particular, Gal4-VP16 is inserted between residues 651 and, 652 ofAPP. The modified APP is generated by means of a mammalian expressionplasmid containing a chimeric gene encoding residues 1-651 of APP, Gal4,VP16, and residues 652-695 of APP (i.e. the cytoplasmic tail, APP ct)under the control of a promoter. The plasmid may further compriseregulatory sequences, linkers, and other elements to facilitate cloning,replication, transfection and expression. A cell comprising the modifiedAPP is provided by transfecting a cell, preferably a mammalian cell, andmost preferably a human cell, with the expression plasmid. The cell iscotransfected with a Gal4 reporter plasmid in which luciferase mRNA isdriven by multiple copies of the Gal4 upstream activating sequence(UAS). When the modified APP is cleaved by γ-secretase, the cleavageproduct containing Gal4-VP16 enters the nucleus and transactivatestranscription from the Gal4 reporter plasmid. Expression of luciferaseis measured by standard assays, for example by measuring luciferaseactivity using a commercially available kit. Luciferase expression is ameasure of transactivation, which is in turn a measure of APP cleavage.

[0030] The transfected cells are contacted with a candidate agent, andluciferase expression is measured in the presence and absence of theagent. An agent that increases or decreases luciferase expression is anagent that affects APP cleavage.

[0031] In another embodiment, the present invention provides a method ofidentifying an agent that affects the cleavage of APP comprisingproviding a cell, wherein the cell contains APP and a protein thatinteracts with the C-terminal cleavage product of APP in the nucleus toactivate transcription, and wherein the protein is modified to allowdetection of nuclear translocation of the C-terminal cytoplasmiccleavage product; contacting the cell with a candidate agent; andmeasuring nuclear localization of the C-terminal cytoplasmic cleavageproduct in the presence and absence of the agent. An agent thatincreases or decreases nuclear localization of the C-terminal cleavageproduct relative to nuclear localization in the absence of the cleavageproduct is defined as an agent that affects cleavage of APP.

[0032] In accordance with the present invention, it has been discoveredthat the C-terminal cytoplasmic cleavage product of APP may interactwith one or more other proteins to activate transcription. Accordingly,cleavage of APP can be detected by modifying a protein that interacts,directly or indirectly, with the cleavage product. Direct interactionrefers to proteins that form a complex with the cleavage product.Indirect action includes proteins that interact with other proteins thatare targets of the cleavage product, and thereby includes, for example,proteins that interact with Fe65 and/or Tip60 in the regulation oftranscription. A preferred protein is the histone acetyl-transferaseTip60. In a preferred embodiment of this method, a protein thatinteracts with the C-terminal cleavage product of APP to activatetranscription, for example Tip60 is modified to allow detection ofnuclear localization, for example by fusion with the DNA binding domainof a transcriptional activator such as Gal4 or LexA. Nuclearlocalization is measured by determining activation of transcription ofan indicator gene that is under the transcriptional control of a bindingsite for the DNA binding domain, as described hereinabove.

[0033] The nucleotide and amino acid sequences of Tip60 are known in theart and disclosed for example by Kamine et al. (1996) Virology 216,357-366 and Ran et al. (2000) Gene 258, 141-146. The term Tip60 as usedherein includes modifications such as insertions, deletions andsubstitutions provided that the ability of Tip60 to interact with theC-terminal cytoplasmic cleavage product of APP is maintained.

[0034] In a preferred embodiment of this method, the cells contain APP,Fe65 and Tip60 modified to contain the DNA binding domain of atranscriptional activator, preferably Gal4. Such cells may be obtainedby co-transfection with plasmids containing nucleic acids encoding APP,Fe65, and modified Tip60. Plasmids may further comprise regulatorysequences, linkers and other elements to facilitate cloning,replication, transfection and expression. Cells are eukaryotic,including for example insect and mammalian, and preferably human. Cellsare also co-transfected with the appropriate reporter plasmid asdescribed above. Expression of the reporter gene is a measure oftransactivation, which is in turn a measure of nuclear localization ofthe C-terminal cytoplasmic cleavage product of APP, and thus APPcleavage. Reporter gene expression is measured as described hereinabove.

[0035] In another embodiment the present invention provides vectors thatcontain nucleic acids encoding the modified APP. In a preferredembodiment, the vector comprises a nucleic acid encoding APP operablylinked to a promoter wherein a nucleic acid module encoding aheterologous DNA binding domain of a transcription factor and atranscriptional activator of the same or a different transcriptionfactor is contained within the portion of the nucleic acid that encodesthe C-terminal cytoplasmic tail of APP. A module “within” the tailincludes embodiments in which the module is at the 5′-end or 3′-end ofthe region encoding the cytoplasmic tail. In a preferred embodiment themodule is Gal4-VP16. The vectors may further comprise regulatorysequences, linkers, and other elements,to facilitate cloning,replication, transfection and expression.

[0036] The present invention further provides vectors that comprise anucleic acid encoding APP operably linked to a promoter wherein anucleic acid encoding a heterologous DNA binding domain of atranscription factor is contained with the portion of the nucleic acidthat encodes the C-terminal cytoplasmic tail of APP. The DNA bindingdomain may be at the 5′-end or 3′-end of the region encoding thecytoplasmic tail. In a preferred embodiment the DNA binding domain isGal4. The vectors may further comprise regulatory sequences, linkers,and other elements to facilitate cloning, replication, transfection andexpression.

[0037] The present invention further provides vectors that comprise anucleic acid encoding Tip60 and the DNA binding domain of atranscription factor wherein the nucleic acid is operably linked to apromoter. In a preferred embodiment the DNA binding domain is Gal4. Thevectors may further comprise regulatory sequences, linkers, and otherelements to facilitate cloning, replication, transfection andexpression.

[0038] The present invention further provides cells containing theforegoing vectors. The cells are eukaryotic, preferably mammalian, andmost preferably human. Cells containing the vectors of the invention maybe obtained by methods known in the art, and may be transiently orstably transfected. The cells may also further contain a correspondingreporter plasmid as described hereinabove.

[0039] In another embodiment, the present invention provides agents thataffect cleavage of APP identified by the method of the presentinvention. Compositions comprising the agents are also provided. Thecompositions may comprise carriers and/or diluents such as solvents,dispersion media, antibacterial and antifungal agents, microcapsules,liposomes, cationic lipid carriers, isotonic and absorption delayingagents and the like, as well as supplementary active ingredients.

[0040] The present invention further provides kits useful foridentifying an agent that affects cleavage of APP. The kits comprise afirst compartment containing cells comprising a vector that encodes amodified APP of the invention. The cells may further contain a reporterplasmid. The kits may further comprise a second compartment containing ameans for measuring expression of an indicator gene contained in thereporter plasmid.

[0041] In another embodiment, the kits comprise a first compartmentcontaining a vector that encodes a modified APP of the invention. Thekits may further comprise a second compartment containing a reporterplasmid, and may further comprise a third compartment containing cellssuitable for transfection by the vector of the first compartment.

[0042] In a further embodiment, the kits comprise a first compartmentcontaining a vector comprising a nucleic acid encoding APP operablylinked to a promoter, a second compartment containing a vectorcomprising a nucleic acid encoding Fe65 operably linked to a promoter,and a third compartment containing a nucleic acid encoding a fusionprotein comprising Tip60 and the DNA binding domain of a transcriptionalactivator, preferably Gal4.

[0043] The following nonlimiting examples serve to further illustratethe present invention.

EXAMPLE I Materials and Methods

[0044] The following materials and methods were used in subsequentexamples.

[0045] 1. Transactivation Assays

[0046] 1.1 General Design

[0047] PC12, COS, HeLa, and HEK293 cells were co-transfected at 50-80%confluency in 6-well plates using Fugene6 (Roche, Indianapolis, Ind.),and 3-4 plasmids (0.1-1.0 μg DNA/well depending on cell types; seeplasmid list below for description of all constructs). All transfectionsincluded a. Gal4 (pG5E1B-luc) or LexA (pL8G5-luc) reporter plasmids; b.constitutively expressed β-galactosidase expression plasmid (pCMV-LacZ)to control for transfection efficiency; and c. the Gal4- or LexA-fusionprotein vectors. Cells were harvested 48 hr post-transfection in 0.2ml/well reporter lysis buffer (Promega, Madison, Wis.), and theirluciferase and β-galactosidase activities were determined with thePromega luciferase assay kit and the standardO-nitrophenyl-D-galacto-pyranoside method, respectively. The luciferaseactivity was standardized by the β-galactosidase activity to control fortransfection efficiency and general effects on transcription, and inmost experiments further normalized for the transactivation observed incells expressing Gal4 or LexA alone. Values shown are averages oftransactivation assays carried out in duplicate, and repeated at leastthree times for each cell type and constructs. Most constructs wereassayed in three or four cell lines, but usually only representativeresults for one cell line are shown. To confirm expression oftransfected proteins and secretase cleavage of the various APPconstructs, transfected cells were also analyzed by immunoblotting usingantibodies to the respective proteins and/or antibodies to the epitopetags attached to the proteins.

[0048] 1.2 Amounts of DNA Used

[0049] 1.2.1. APP-Gal4VP16 Assays

[0050] Plasmids used in the following amounts: a. pG5E1B-luc (HEK293cells, HeLa cells, and COS cells=0.3-0.5 μg DNA; PC12 cells=1.0 μg); b.pCMV-LacZ (HEK293 cells, HeLa cells, and COS cells=0.05 μg DNA; PC12cells=0.5 μg DNA); c. pMst (Gal4), pMst-GV-APP (APP-GV), pMst-GV (GV),pMst-GV-APPct (APPct-GV), pMst-APPct (APPct-Gal4), pMst-GV-APP*(APP*-GV), pMst-GV-APPγ* (APPγ*-GV), pMst-APPct* (APPct*-Gal4),pMst-GV-APPα (APPα-GV), pMst-GV-NRX (NRX-GV), pMst-GV-NA (NRXe-GV-APPc),pMst-GV-AN (APPe-GV-NRXc)(HEK293, HeLa, and COS cells=0.1-0.3 μg DNA;PC12 cells=1.0 μg DNA). d. pcDNA3.1-PS2D366A, pCMV-Mint1, or pCMV5-Fe65(HEK293, HeLa, and COS cells=0.1-0.3 μg DNA; PC12 cells=0.5 μg DNA).

[0051] 1.2.2. APP-Gal4 and APP-LexA assays with and without various Fe65and Mint1 Constructs

[0052] For transactivation by APP-Gal4 and APP-LexA constructs, cellswere cotransfected with: a. pG5E1B-luc (Gal4 reporter plasmid) orpL8G5-luc (LexA-reporter plasmid)(HEK293 cells, HeLa cells, and COScells=0.3 μg DNA; PC12 cells=1.0 μg); b. pCMV-LacZ (β-galactosidasecontrol plasmid. HEK293 cells, HeLa cells, and COS cells=0.05 μg DNA;PC12 cells=0.2-0.5 μg DNA); c. pMst (Gal4), pMst-APP (APP-Gal4),pMst-APP* (APP*-Gal4), pMst-APPγ (APPγ-Gal4), pMst-APPγ* (APPγ*-Gal4),pMst-AN-APPc32 (APP-G-NRX-APPc32), pMst-AN (APPe-G-NRXc), pML (LexA),pML-APP (APP-LexA), pML-APP* (APP*-LexA), pML-APPct (APPct-LexA),pML-APPct* (APPct*-LexA) (HEK293 cells, HeLa cells, and COScells=0.3-0.5 μg DNA; PC12 cells=1.0-1.5 μg); and d. pCMV-Mint1 (mint1)or pCMV5-Fe65 (Fe65) (HEK293 cells, HeLa cells, and COS cells=0.3-0.5 μgDNA; PC12 cells=1.0-1.5 μg) where indicated.

[0053] For transactivation assays of Fe65 mutants, cells werecotransfected with: a. pG5E1B-luc (Gal4 reporter plasmid) or pL8G5-luc(LexA-reporter plasmid); b. pCMV-LacZ (β-galactosidase control plasmid);c. pMst (Gal4), pMst-APP (APP-Gal4), pML (LexA), or pML-APP (APP-LexA);and d. pCMV5-Fe65 (Fe65), pCMVMyc-Fe65(128-711) (Fe65(128-711)),pCMVMyc-Fe65(242-711) (Fe65(242-711)), pCMVMyc-Fe65(287-711)(Fe65(287-711)), pCMV5-Fe65(1-553) (Fe65□PTB2), pCMVMyc-Fe65ΔPTB1(Fe65ΔPTB1), pCMV5-Fe65mW1 (Fe65mW1), pCMV5-Fe65mW2 (Fe65mW2),pCMV5-Fe65mW3 (Fe65mW3), pCMV5-Fe65mW4 (Fe65mW4), or pCMV5-Fe65mW5(Fe65mW5) where indicated. a. and b.: amounts of DNA same as under 1.21.c. and d.:0.3-0.5 μg DNA for COS, HeLa, and HEK293 cells; 1.0-1.5 μg DNAfor PC12 cells.

[0054] 1.2.3. Gal4-Tip60 Assays

[0055] For transactivation assays of Gal4-Tip60, COS and HEK293 cellswere cotransfected with: a. pG5E1B-luc (Gal4 reporter plasmid, 0.3 μgDNA); b. pCMV-LacZ (β-galactosidase control plasmid, 0.05 μg DNA); c.pMst (Gal4); pM-Tip60 (rat Gal4-Tip60 β residues 63-454); pM-Tip60*(mutant rat Gal4-Tip60 β residues 63-454); pM-hTip60 (full-length wildtype human Gal4-Tip60 β); or pM-hTip60* (full-length mutant humanGal4-Tip60 β) (0.4 μg DNA) d. pCMV5-Fe65 (Fe65), pCMVMyc-Fe65(242-711)(Fe65(242-711)), pCMV5-Fe65(1-553) (Fe65)PTB2), or pCMV5-Fe65mW4(Fe65mW4) (0.3 μg DNA) where indicated; and e. pCMV5-APP (human APP695)or pCMV5-APP* (mutant human APP695) (0.3 μg DNA). All transfectionscontained one of the plasmids listed in a-c, whereas d and e werevariable.

[0056] 2. Yeast Two-hybrid Screens for APP- and Fe65-binding Proteins

[0057] 2.1 APP Screens

[0058] Bait: pBTM116-APP

[0059] Yeast strain: L40

[0060] Library: P8 rat brain library constructed in prey vector pVP16-3.

[0061] Screening condition: 250 ml mid-log phase yeast harboring thebait vector pBTM116-APP were transformed with 125 μg of P8 rat brainlibrary plasmids, and plated on CSM-Trp-Leu-His plates supplemented with5 mM 3-amino-1,2,4-triazole. 2×10⁷ total transformants were obtained.Around 250 positives appeared after 3 days incubation at 30° C. 80positives were recovered and identified by sequencing or Southernblotting. Among them, 72 are Fe65 and one is Fe65-like protein 2(Fe65LP2).

[0062] Summary of Fe65 prey clones that were sequenced Prey ClonesInsert Size (kb) Residues Extra Residues P29^(a) 3.1  1-711 43 P50, P64,P65, P69 2.9  1-711 43 P42 3.0  48-711 0 P60^(a) 2.6 128-711 0 P6^(a),P7, P18 1.9 242-711 8 P57 2.3 242-711 50 P19, P25^(b) 1.0 242-?  21 P15,P27^(a) 1.6 287-711 0 P21 1.5 301-711 0 P4, P22 1.4 313-711 0 P9, P16,P17 1.9 321-711 0 P39 1.4 339-711 0

[0063] 2.2 Fe65 Screens

[0064] Bait: pLexN-Fe65(287-711)

[0065] Yeast strain: L40

[0066] Library: P8 rat brain library constructed in prey vector pVP16-3.

[0067] Screening condition: 250 ml mid-log phase yeast harboring thebait vector pLexN-Fe65(287-711) were transformed with 125 μg of P8 ratbrain library plasmids, and plated on CSM-Trp-Leu-His platessupplemented with 25 mM 3-amino-1,2,4-triazole. 7×10⁷ totaltransformants were obtained, and ˜300 positives selected after 3 daysincubation at 30° C.

[0068] Summary of prey clones that were sequenced: Tip60 β (residues63-454)=8 clones; APLP1=9 clones.

[0069] 3. Quantitative Yeast Two-hybrid Assays

[0070] For quantitative measurements of yeast two-hybrid interactions,various bait and prey plasmids were co-transfected into yeast strain L40and plated on CSM-Trp-Leu (Bio101) plates. Single colonies from theCSM-Trp-Leu plates were cultured in CSM-Trp-Leu liquid medium after 3days, and re-inoculated into YPAD medium at OD₆₀₀=0.3 on the followingday. When the yeast cultures reached mid-log phase (OD₆₀₀=0.6-1.0),cells were collected by centrifugation, washed in Z buffer (16.1 g/lNa₂HPO₄.7H₂O, 5.5 g/l NaH₂PO₄.H₂O, 0.75 g/l KCl, 0.246 g/l MgSO₄.7H₂O pH7.0), and resuspended in 100 μl Z buffer. Cells were lysed by threefreeze-thawing cycles, 0.8 ml reaction solution (0.5 g/l ONPG and 0.024%β-mercaptoethanol in Z buffer) was added, and reactions were stoppedwith 0.4 ml 1M Na₂CO₃ at the appropriate time points depending on colordevelopment. After centrifugation (14,000×g for 10 min), OD₄₂₀ wasmeasured, and the relative β-galactosidase activity was calculated as1000×OD₄₂₀/(min×vol. yeast in ml×OD₆₀₀).

[0071] 3.1 APP & Fe65 Time ∃-gal # Bait Prey OD₆₀₀ (min) OD₄₂₀ unit MeanS.D. 1 APPct P29(Fe65 1-711) 0.899 15 5.025 372.6363 423.8138 48.305290.764 15 4.93 430.192 0.685 15 4.815 468.6131 2 APPct Fe65mW5 0.657 154.895 496.7022 449.4991 71.701238 0.68 15 4.945 484.8039 0.822 15 4.525366.9911 3 APPct vector 0.813 15 0.004 0.328003 0.370065 0.0425321 0.90315 0.005 0.36914 0.807 15 0.005 0.413052 4 APPct* P29(Fe65 1-711) 0.77615 0.005 0.429553 0.506697 0.0674611 0.601 15 0.005 0.554631 0.622 150.005 0.535906 5 APPct* Fe65mW5 0.631 15 0.004 0.42261 0.44523 0.04676280.644 15 0.004 0.414079 0.668 15 0.005 0.499002

[0072] 3.2 Fe65 & LBP-1c Time ∃-gal # Bait Prey OD₆₀₀ (min) OD₄₂₀ unitMean S.D. 1 Fe65(287-711) LBP-1c 0.747 60 0.037 0.825524 0.7723760.1010779 0.737 60 0.029 0.655812 0.678 60 0.034 0.835792 2Fe65(287-531) LBP-1c 0.686 60 0.167 4.057337 4.438847 0.3744772 0.704 600.203 4.805871 0.625 60 0.167 4.453333 3 Fe65(287-711) vector 0.709 600.006 0.141044 0.15965 0.0262405 0.703 60 0.008 0.189663 0.787 60 0.0070.148242 4 Fe65(287-531) vector 0.725 60 0.022 0.505747 0.5543 0.07143020.681 60 0.026 0.636319 0.672 60 0.021 0.520833 5 Lamin LBP-1c 0.626 600.017 0.452609 0.721477 0.3382954 0.819 60 0.03 0.610501 0.454 60 0.031.101322

[0073] 3.3 Fe65 & hTip60 Time ∃-gal # Bait Prey OD₆₀₀ (min) OD₄₂₀ unitMean S.D. 1 Fe65(287-711) hTip60 0.806 15 4.905 405.7072 431.792745.395309 0.818 15 4.975 405.4605 0.665 15 4.83 484.2105 2 Fe65(287-531)hTip60 0.695 15 3.895 373.6211 391.1748 52.914479 0.774 15 4.055349.2679 0.682 15 4.61 450.6354 3 Fe65(287-711) vector 0.709 60 0.0060.141044 0.15965 0.0262405 0.703 60 0.008 0.189663 0.787 60 0.0070.148242 4 Fe65(287-531) vector 0.725 60 0.022 0.505747 0.5543 0.07143020.681 60 0.026 0.636319 0.672 60 0.021 0.520833 5 Lamin hTip60 0.594 150.011 1.234568 1.751572 0.5012274 0.635 15 0.017 1.784777 0.507 15 0.0172.235371

[0074] 4. Plasmid list

[0075] 4.1 Standard plasmids

[0076] pGMV-LacZ: Transfection control plasmid encoding bacterialβ-galactosidase under control of the CMV promoter.

[0077] pG5E1B-luc: Gal4 reporter plasmid (Lillie, J. W., and M. R.Green. 1989. Transcription activation by the adenovirus E1a protein.Nature (London) 338:39-44) in which luciferase mRNA is driven by fivecopies of Gal4 UAS.

[0078] pL8G5-luc: LexA reporter plasmid in which luciferase mRNA isdriven by eight copies of the LexA binding site and five copies of GalUAS. (Hollenberg, S. M., Sternglanz, R., Cheng, P. F., and Weintraub H.1995. Identification of a new family of tissue-specific basicHelix-Loop-Helix proteins with a two-hybrid system. Mol. Cell. Biol.15:3813-3822).

[0079] pMst: Gal4 expression vector driven by the SV40 promoter derivedfrom pM (Clontech, Palo Alto, Calif.) by mutating the stop codon beforethe Gal4 DNA-binding domain.

[0080] pMst-GV: Gal4 VP16 (GV) expression vector generated by cloningthe VP16 activation domain (residues 413-490) into the EcoRI/BamHI sitesof pMst (linker sequence between Gal4 and VP16: QLTVSPEFAPPTD).

[0081] pML: LexA expression vector generated by replacing the NheI/EcoRIfragment of pM (Clontech) with the PCR amplified LexA-coding sequence.

[0082] 4.2 APP Plasmids

[0083] 4.2.1. Mammalian Expression Plasmids

[0084] pMst-GV-APPct, encodes APPct-GV generated by cloning thecytoplasmic tail of human APP695 (APPct, residues 652-695) into theBamHI/SalI sites of pMst-GV (linker sequence between GV andAPPct=DEYGGGIPPGQYTSI).

[0085] pMst-GV-APPct*, encodes APPct*-GV with point mutations in thecytoplasmic tail of APP (residues 684-687; wild type sequence=NPTY;mutant sequence=NATA). Generated by QuickChange site-directedmutagenesis (Stratagene) with pMst-GV-APPct as template.

[0086] pMst-GV-APP encodes APP-GV. Generated by cloning a PCR fragmentencoding residues 1-651 of human APP695 into the NheI site ofpMst-GV-APPct (linker sequence between APPe and GV=MLKKKPLASSRMKLLS).

[0087] pMst-GV-APP*, encodes APP*-GV with point mutations in thecytoplasmic tail of APP (residues 684-687; wild type sequence=NPTY;mutant sequence=NATA). Generated by QuickChange site-directedmutagenesis (Stratagene) with pMst-GV-APP as the template.

[0088] pMst-GV-APPγ, encodes APPγ-GV containing an N-terminal methioninefollowed by residues 639-651 of human APP695, Gal4-VP16, and residues652-695 of APP695. Obtained by inserting the PCR-amplified residues639-651 into the BglII/NheI sites of pMst-GV-APPct (linker sequencebetween TMR of APP and Gal4=MLKKKPLASSRMKLLS).

[0089] pMst-GV-APPγ*, encodes APPγ*-GV corresponding to APPγ-GV with themutation in the NPTY sequence. Generated by QuickChange site-directedmutagenesis (Stratagene) with pMst-GV-APPγ as the template.

[0090] pMst-APPct, encodes APPct-Gal4. Generated by cloning thecytoplasmic tail of human APP695 (APPct, residues 652-695) into theBamHI/SalI sites of pMst (linker sequence between Gal4 andAPPct=QLTVSPEFPGIPPGQYTSI).

[0091] pMst-APPct*, encodes APPct*-Gal4 corresponding to APPct-Gal4 withthe NPTY mutation. Generated by cloning the mutant cytoplasmic tail frompMst-GV-APPct* into the BamHI/SalI sites of pMst.

[0092] pMst-APP, encodes APP-Gal4. Generated by cloning a PCR fragmentcontaining the extracellular and transmembrane region of human APP695(APPe, residues 1-651) into the NheI site of pMst-APPct (linker sequencebetween APPe and Gal4=MLKKKPLASSRMKLLS).

[0093] pMst-APP*, encodes APP*-Gal4. Obtained as pMst-APP, but clonedinto the NheI site of pMst-APPct*.

[0094] pMst-APPγ, encodes APPγ-Gal4. Generated by cloning a PCR fragmentcoding for a methionine and residues 639-651 of human APP695 into theBglII/NheI sites of pMst-APPct (linker sequence between TMR andGal4=MLKKKPLASSRMKLLS).

[0095] pMst-APPγ*, encodes APPγ*-Gal4. Obtained as pMst-APPγ, but clonedinto pMst-APPct*.

[0096] pMst-GV-NRX, encodes NRX-GV. Generated by sequential cloning ofPCR fragments coding for the cytoplasmic tail of rat neurexin I (βNRXct,residues 414-468) and the extracellular and transmembrane regions (NRXe,residues 1-417) into the BamHI/SalI and the NheI sites, respectively, ofpMst-GV (linker sequences between NRXe and Gal4=MYKYRTLASSRMKLLS;between VP16 and NRXct=DEYGGGIPPGYKYRN).

[0097] pMst-GV-NA, encodes NRXe-GV-APPc. Generated by cloning a PCRfragment corresponding to NRXe into the NheI site of pMst-GV-APPct(linker sequence between NRXe and Gal4=MYKYRTLASSRMKLLS).

[0098] pMst-GV-AN, encodes APPe-GV-NRXc. Generated by sequential cloningof PCR fragments corresponding to NRXc and APPe into the BamHI/SalI andthe NheI sites, respectively, of pMst-GV (linker sequences between APPeand Gal4=MLKKKPLASSRMKLLS; between VP16 and NRXct=DEYGGGIPPGYKYRN).

[0099] pMst-AN encodes APPe-G-NRXc. Generated as pMst-GV-AN, but intopMst (linker sequences between APPe and Gal4=MLKKKPLASSRMKLLS, andbetween Gal4 and NRXct=QLTVSPEFPGIPPGYKYRN.

[0100] pMst-AN-APPc32, encodes APP-G-NRX-APPc32. Generated from pMst-ANby inserting a PCR fragment encoding the C-terminal 32 residues of humanAPP695 (APPc32) into the rat neurexin I cytoplasmic tail (NRXct) betweenresidue 425 and 427, with V426 deleted during the cloning (linkersequences between APPc32 and NRXct=EGSYHIDDAAVT, and between APPc32 andNRXct=EQMQNIDESRN.

[0101] pML-APPct, encodes APPct-LexA. Generated by replacing the Gal4sequence (the NheI/EcoRI fragment) in pMst-APPct with the LexA-sequence(linker sequence between LexA and APPct=NGDWLEFPGIPPGQYTSI).

[0102] pML-APPct*, encodes APPct*-LexA. Generated as pML-APPct inpMst-APPct*.

[0103] pML-APP, encodes APP-LexA. Generated by cloning the extracellularand transmembrane region of human APP695 (APPe, residues 1-651) into theNheI site of pML-APPct (linker sequence between APPe andLexA=MLKKKPLAKMKALT).

[0104] pML-APP*, encodes APP*-LexA. Generated as pML-APP withpML-APPct*.

[0105] pCMV5-APP, encodes full-length human APP695 inserted into theblunted-EcoRI/XbaI sites of pCMV5.

[0106] pCMV5-APP*, encodes full length human APP695 containing pointmutations in the cytoplasmic NPTY sequence. Generated by QuickChangesite-directed mutagenesis (Stratagene) with pCMV5-APP.

[0107] 4.2.2. Yeast Two-hybrid Plasmids

[0108] pBTM116-APP, encodes residues 648-695 of human APP695 cloned intothe BamHI/SalI sites of the yeast two-hybrid bait vector pBTM116 using aPCR fragment.

[0109] pBTM116-APP*=pBTM116-APP in which the codons encoding the NPTYsequence in the APP cytoplasmic tail were mutated to NATA usingQuickChange site-directed mutagenesis (Stratagene).

[0110] 4.3 Fe65 Plasmids

[0111] 4.3.1. Mammalian Expression Plasmids

[0112] pCMV5-Fe65: encodes full-length rat Fe65 (711 residues).Constructed by sub-cloning the 3 kb SalI fragment from the yeasttwo-hybrid prey clone #P29 into the SalI site of pCMV5.

[0113] pCMVMyc-Fe65(128-711): encodes residues 128-711 of Fe65.Generated by cloning the blunt-ended . . . fragment from the rat Fe65cDNA into the blunted EcoRI site in pCMVMyc.

[0114] pCMVMyc-Fe65(242-711): encodes residues 242-711 of Fe65.Generated by cloning the blunt-ended . . . fragment from the rat Fe65cDNA into the blunted EcoRI site in pCMVMyc.

[0115] pCMVMyc-Fe65(287-711): encodes residues 287-711 of Fe65.Generated by cloning the blunt-ended . . . fragment from the rat Fe65cDNA into the blunted EcoRI site in pCMVMyc.

[0116] pCMV5-Fe65(1-553): encodes Fe65 PTB2 which lacks residues 554-711of Fe65. Generated by introducing a stop codon into pCMV5-Fe65 afterresidue 553 with the QuickChange site directed mutagenesis kit(Stratagene).

[0117] pCMVMyc-Fe65 ΔPTB1: encodes Fe65 ΔPTB1 which lacks residues314-440. Generated by sequentially cloning the PCR fragments encodingresidues 441-711 and residues 1-313 of rat Fe65 into the ClaI and MluIsites, respectively, of pCMVMyc.

[0118] pCMV5-Fe65mW1: encodes Fe65mW1 point mutant in carryingsubstitions W281F and P284A. Generated by QuickChange site directedmutagenesis (Stratagene) with pCMV5-Fe65 as template.

[0119] pCMV5-Fe65mW2: encodes Fe65mW2 point mutant in carryingsubstiution W260F. Generated by QuickChange site directed mutagenesis(Stratagene) with pCMV5-Fe65 as template.

[0120] pCMV5-Fe65mW3: encodes Fe65mW3 point mutant in carryingsubstitutions W260F, W281F and P284A. Generated by QuickChange sitedirected mutagenesis (Stratagene) with pCMV5-Fe65mW1 as template.

[0121] pCMV5-Fe65mW4: encodes Fe65mW4 point mutant in carryingsubstitutions Y270A, Y271A, and W272A. The insert (2.1 kb) can be cutout by HindIII+SalI double digestion.

[0122] pCMV5-Fe65mW5: encodes Fe65mW4 point mutant in carryingsubstitutions Y270A, Y271A, W272A, W281F, and P284A. The insert (2.1 kb)can be cut out by HindIII+SalI double digestion.

[0123] pcDNA3.1-N-HA-Fe65: encodes full-length rat Fe65 preceded by ahemagglutinin (HA) epitope. Obtained by subcloning the rat Fe65 cDNAinto the blunted-EcoRI/XbaI sites of pcDNA3.1-N-HA.

[0124] 4.3.2. Yeast Two-hybrid Plasmids

[0125] pLexN-Fe65(287-711), encodes residues 287-711 of Fe65 in theSalI/blunted-PstI sites in pLexN.

[0126] pLexN-Fe65(287-531), encodes residues 287-531 of Fe65 in theBamHI/blunted-SalI sites in pLexN. Insert (740 bp) can be cut out byBamHI+PstI double digestion.

[0127] pVP16-3-Fe65mW5, encodes the mW5 mutant of Fe65 (see pCMV vectorsabove). Was generated by cloning the blunted 2.1 kb HindIII/XbaIfragment from pCMV5-Fe65mW5 into the blunted-NotI/XbaI sites of theyeast prey vector pVP16-3. The insert can be cut out by SalI.

[0128] 4.4 Tip60 Plasmids

[0129] 4.4.1. pCMV Expression Plasmids

[0130] pCMVMyc-Tip60 (63-454 , encodes residues 63-454 of rat Tip60β.Generated by cloning the 1.3 kb EcoRI fragment from yeast two-hybridprey clone #B36 into the EcoRI site of pCMVMyc.

[0131] pCMVMyc-Tip60(63-454)*, encodes residues 63-454 of rat Tip60βwith a mutation in residues 257-260 (sequences: wildtype=NKSY;mutant=NASA). Generated by QuickChange site directed mutagenesis kit(Stratagene) with pCMVMyc-Tip60(63-454) as template.

[0132] pM-Tip60 encodes rat Tip60β residues 63-454 preceded by the Gal4DNA-binding domain. Generated by subcloning the 1.3 kb BamHI/XbaIfragment from prey clone #36 into the BamHI/XbaI sites of pM.

[0133] pM-Tip60* encodes the same protein as pM-Tip60 with theinactivating mutation in residues 257-260. Generated by cloning the 1.3kb BamHI/XbaI fragment from pVP16-3-Tip60* into the BamHI/XbaI sites ofpM.

[0134] pCMVMyc-hTip60 encodes myc-tagged full-length human Tip60β.Obtained by subcloning the insert of EST IMAGE clone 2901054 into theMluI/XbaI sites of pCMVMyc.

[0135] pM-hTip60 encodes full-length human Tip60β preceded by theGal4-DNA binding domain. Obtained by cloning the blunted 1.6 kbEcoRI/NotI fragment from EST clone 2901054 into the blunted EcoRI siteof pM. Insert can be cut out by SalI.

[0136] pM-hTip60*, same as pM-hTip60 but with the inactivating mutationin residues 257-260. Generated by QuickChange site directed mutagenesis(Stratagene) with pM-hTip 60 as template.

[0137] 4.5 Yeast Two-hybrid Plasmids

[0138] Prey clone #B36 in pVP16-3; identified in yeast two-hybridscreens with pLexN-Fe65(287-711) as the bait in a P8 rat brain library.B36 encodes rat Tip60β corresponding to residues 63-454 of human Tip60,with a single amino acid change between human and rat sequences.

[0139] pVP16-3-Tip60* encoding mutant rat Tip60 with the inactivatingmutation in residues 257-260 (sequences: wildtype=NKSY; mutant=NASA).Generated by cloning the EcoRI fragment from pCMVMyc-Tip60(63-454)* intothe EcoRI site of yeast prey vector pVP16-3.

[0140] pVP16-3-hTip60, full-length human Tip60 cloned into theEcoRI/NotI sites of P16-3.

[0141] 4.6 GST-fusion Protein Plasmids

[0142] pGEX-KG-Tip60 (63-454), residues 63-454 Tip60β fused to GST.Generated by cloning the 1.3 kb EcoRI fragment (1.3 kb) of the yeasttwo-hybrid prey clone B36 into the EcoRI site of pGEX-KG.

[0143] pGEX-KG-Tip60(63-454)*, encodes residues 63 454 Tip60β fused toGST. Generated as pGEX-KG-Tip60 (63-454), but frompVP16-3-Tip60(63-454)*.

[0144] 5. Miscellaneous Plasmids

[0145] 5.1 pCMV Expression Plasmids

[0146] pCMV5-Mint-1: rat Mint1 cloned into the EcoRI site of pCMV5(Okamoto, M. and Südhof, T. C. (1997) J. Biol. Chem. 272, 31459-31464.)

[0147] pCS2+MT-SEF: Myc-tagged full-length human LBP-1c (1-450 residues)was expressed from control of the CMV promoter (gift from Dr. W. S. L.Liao, University of Texas MD Anderson Cancer Center, Houston Tex.;reference: Z. Bing, S. A. G. Reddy, Y. Ren, J. Qin, and W. S. L. Liao(1999) J. Biol. Chem. 274, 24649-24656.)

[0148] pcDNA3.1-PS2D366A (kind gift of Dr. C. Haass, Munich): encodes adominant negative mutant of human presenilin 2 in pcDNA3.1.

[0149] 5.2 Yeast Two-hybrid Plasmids

[0150] pVP16-3-LBP-1c, encodes full-length human LBP-1c. Generated bycloning the blunted 1.4 kb XhoI fragment from pCS2+MT-SEF into theblunted NotI site of the yeast prey vector pVP16-3. Insert can by cutout by XhoI.

[0151] 6. Transfections, Transactivation Assays, and Yeast Two HybridScreens

[0152] PC12, COS, HeLa, and HEK293 cells were co-transfected at 50-80%confluency in 6-well plates using Fugene6 (Roche), and 3-5 plasmids(0.1-1.0 μg DNA/well depending on cell types; see plasmid listhereinabove for description of all constructs). All transfectionsincluded a. Gal4 (pG5E1B-luc) or LexA (pL8G5-luc) reporter plasmids; b.constitutively expressed β-galactosidase expression plasmid (pCMV-LacZ)to control for transfection efficiency; and c. the Gal4- or LexA-fusionprotein vectors. Cells were harvested 48 hr post-transfection in 0.2ml/well reporter lysis buffer (Promega), and their luciferase andβ-galactosidase activities were determined with the Promega luciferaseassay kit and the O-nitrophenyl-D-galacto-pyranoside method,respectively. The luciferase activity was standardized by theβ-galactosidase activity to control for transfection efficiency andgeneral effects on transcription, and in most experiments normalized forthe transactivation observed in cells expressing Gal4 or LexA alone.Values shown are averages of transactivation assays carried out induplicate, and repeated at least three times for each cell type andconstructs. Most constructs were assayed in three or four cell lines,but usually only representative results for one cell line are shown. Toconfirm expression of transfected proteins and secretase cleavage of thevarious APP constructs, transfected cells were also analyzed byimmunoblotting using antibodies to the respective proteins and/orantibodies to the epitope tags attached to the proteins.

[0153] For assays of the transactivation by Gal4.VP16 constructs, cellswere cotransfected with a. pG5E1B-luc (Gal4 reporter plasmid); b.pCMV-LacZ (β-galactosidase control plasmid); c. pMst (Gal4), pMst-GV-APP(APP-GV), pMst-GV (GV), pMst-GV-APPct (APPct-GV), pMst-APPct(APPct-Gal4), pMst-GV-APP* (APP*-GV), pMst-GV-APPct* (APPct*-GV),pMst-APPct* (APPct*-Gal4), pMst-GV-APPγ (APPγ-GV), pMst-GV-NRX (NRX-GV),pMst-GV-NA (NRXe-GV-APPc), or pMst-GV-AN; and d. pcDNA3.1-PS2D366A (kindgift of Dr. C. Haass, Munich), pCMV-Mint1; or pCMV5-Fe65 whereindicated.

[0154] A yeast two-hybrid cDNA library in pVP16-3 was screened withpBTM116-APP encoding the cytoplasmic tail of human APP₆₉₅ as described(Vojtek et al. (1993) Cell 74, 205-214; Okamoto et al. (1997) J. Biol.Chem. 272: 31459-31464). Of 80 positive clones, 72 encoded Fe65 and oneFe65-like protein. The full-length rat Fe65 sequence has been submittedto GenBank (Acc.# AF333983). Interactions of all proteins includingmutants of Fe65 were quantified using liquid β-galactosidase assays onyeast strains harboring various bait and prey clones (see Example 1).

[0155] For transactivation by APP-Gal4 and APP-LexA constructs, cellswere cotransfected with: a. pG5E1B-luc (Gal4 reporter plasmid) orpL8G5-luc (LexA-reporter plasmid); b. pCMV-LacZ (β-galactosidase controlplasmid); c. pMst (Gal4), pMst-APP (APP-Gal4), pMst-APP* (APP*-Gal4),pMst-APPγ (APPγ-Gal4), pMst-APPγ* (APPγ*-Gal4), pMst-AN-APPc32(APP-G-NRX-APPc32), pMst-AN (APPe-G-NRXc), pML (LexA), pML-APP(APP-LexA), pML-APP* (APP*-LexA), pML-APPct (APPct-LexA), pML-APPct*(APPct*-LexA); and d. pCMV-Mint1 (mint1) or pCMV5-Fe65 (Fe65) whereindicated. Analyses were performed as described above.

[0156] COS7 cells were transfected in 100 mm dishes using DEAE-dextranor Fugene6 (Roche) with single or combinations of expression vectorsencoding wild-type and mutant APPct-Gal4, APPγ-Gal4, and APP-Gal4,myc-tagged or HA-tagged wild type or mutant Fe65, and wild type andmutant Tip60 (see above for a description of the expression vectors),and harvested 72 hr after transfection. For the immunoblottingexperiments (FIG. 3), cell extracts were immunoblotted with polyclonalantibodies to the C-terminus of APP (U955) or to Fe65, and withmonoclonal antibodies to Gal4 (Clontech) or to the myc- or HA-epitope(Santa Cruz). For the immunoprecipitation experiments (FIG. 5), cellswere washed twice with cold PBS, harvested in 1 ml lysis buffer (50 mMHEPES-NaOH pH 7.5, 150 mM NaCl, 10% glycerol, 1% IGEPAL CA-630, 1.5 mMMgCl12, 1 mM EGTA, 1 mM DTT, 0.1 g/L PMSF, 10 mg/L Leupeptin, 10 mg/Laprotinin, 1 mg/L pepstatin A), and passed through a 28 gauge needle 5×.Cell extracts were clarified by centrifugation at 20,800×g for 10 min.The supernatants (˜1 ml) were incubated with 10 μl of a polyclonalantibody raised against the C-terminus of APP (U955) or monoclonalantibodies to myc-tag (Santa Cruz) for 2 hr at 4° C., 60 μl of a 50%slurry of protein A- or protein G-Sepharose (Phamacia) were added, andthe beads were incubated with the reactions for 1 hr at 4° C. on arotator and then collected by centrifugation. Beads were washed 3× withlysis buffer, resuspended in 0.1 ml SDS-PAGE sample buffer, and 20 μl ofthe protein solutions were resolved on 10% SDS-PAGE, and detected byimmunoblotting with antibodies to APP, Gal4, or the myc-epitope.

[0157] For transactivation assays of Fe65 mutants, cells werecotransfected with: a. pG5E1B-luc (Gal4 reporter plasmid) or pL8G5-luc(LexA-reporter plasmid); b. pCMV-LacZ (β-galactosidase control plasmid);c. pMst (Gal4), pMst-APP (APP-Gal4), pML (LexA), or pML-APP (APP-LexA);and d. pCMV5-Fe65 (Fe65), pCMVMyc-Fe65(128-711) (Fe65(128-711)),pCMVMyc-Fe65(242-7 11) (Fe65(242-711)), pCMVMyc-Fe65(287-711)(Fe65(287-711)), pCMV5-Fe65(1-553) (Fe65 PTB2), pCMVMyc-Fe65ΔPTB1(Fe65ΔPTB1), pCMV5-Fe65mW1(Fe65mW1), pCMV5-Fe65mW2 (Fe65mW2),pCMV5-Fe65mW3 (Fe65mW3), pCMV5-Fe65mW4 (Fe65mW4), or pCMV5-Fe65mW5(Fe65mW5) where indicated. Analyses were performed as described above,and plasmids are described above.

[0158] Yeast two-hybrid screens were carried out with a fragment fromrat Fe65 (residues 287-711) as described above. Out of 100 clonesanalyzed, 9 clones encoded APLP1, and 8 clones Tip60β (residues 63-454of the insert-minus splice β-variant; submitted to GenBank with Acc.#AF333984). The domains of Fe65 that bind to the cytoplasmic tail of APPor to Tip60 were studied by quantitative yeast two-hybrid assays whichdemonstrated that the first PTB domain of Fe65 is necessary andsufficient for binding to Tip60, and the second PTB domain for bindingto APP. For Tip60, both the partial rat cDNA and the full-length humancDNA were analyzed (see FIG. 6A).

[0159] GST-pulldowns were performed essentially as described by Hata etal. (1993) Nature 366, 347-351 using purified wild type and mutant ratGST-Tip60β and Fe65 expressed by transfection in COS cells. Extractsfrom transfected COS cells were preabsorbed with 10 μg GST onglutathione agarose for 2 hr at 4° C., and then incubated for 4 hrs at4° C. with 10 μg of GST-Tip60, GST-Tip60*, or GST bound to glutathioneagarose. Beads were washed 5× in lysis buffer, resuspended in 80 μlSDS-PAGE sample buffer, and 20 μl were analyzed by SDS-PAGE andimmunoblotting using antibodies to Fe65 and to the myc epitope.Co-immunoprecipitation experiments of Fe65, APP, and wild-type andmutant rat Tip60β were performed as described above using COS cellsco-transfected with the appropriate vectors.

[0160] HeLa cells plated on cover glass in a 12-well plate weretransfected with pcDNA3.1-N-HA-Fe65 and pCMVMyc-hTip60 (0.25 μg for eachplasmid) using Fugene6 (Roche). Two days after transfection, cells werewashed twice with PBS, fixed (3.7% formaldehyde for 10 min at roomtemperature), and blocked and permeabilized in PBS containing 3% BSA,0.1% IGEPAL CA-630 for 20 min. Cells were then incubated with anti-HAmonoclonal antibody (BAbCO Berkeley antibody company) and anti-Mycpolyclonal antibody (Upstate Biotechnology) for 1 hr (1:200 dilution inblocking buffer), washed with PBS 3×, and treated withRhodamine-goat-anti-mouse and FITC-goat-anti-rabbit antibodies(Chemicom) for 1 hr (1:500 dilution in blocking buffer). After 3 washeswith PBS and one wash with water, cells were mounted and observed with aconfocal microscope.

[0161] For transactivation assays of Gal4Tip60, COS and HEK293 cellswere cotransfected with: a. pG5E1B-luc (Gal4 reporter plasmid); b.pCMV-LacZ (β-galactosidase control plasmid); c. pMst (Gal4); pM-Tip60(rat Gal4-Tip60β residues 63-454); pM-Tip60* (mutant rat Gal4-Tip60βresidues 63-454); pM-hTip60 (full-length wild type human Gal4-Tip60β);or pM-hTip60* (full-length mutant human Gal4-Tip60β) d. pCMV5-Fe65(Fe65), pCMVMyc-Fe65(242-711) (Fe65(242-711)), pCMV5-Fe65(1-553) (Fe65PTB2), or pCMV5-Fe65mW4 (Fe65mW4) where indicated; and e. pCMV5-APP(human APP695) or pCMV5-APP* (mutant human APP695). All transfectionscontained one of the plasmids listed in a-c, whereas d and e werevariable. Analyses were performed as described above.

EXAMPLE 2

[0162] Nuclear signaling by the cytoplasmic γ-cleavage product of APPfused to Gal4.VP16. Cleavage of APP produces a C-terminal fragmentcomposed of half of the TMR (10-12 residues) and the cytoplasmic tail(47 residues) (See, e.g. Selkoe (1998) Trends Cell Biol. 8, 447-453). Atranscription factor was engineered into the cytoplasmic tail of APP.The inserted transcription factor was Gal4.VP16 which is composed of ayeast DNA-binding protein (Gal4) fused to a powerful viral activator(VP16) (Sadowski et al. (1988) Nature 335, 563-564). When inserted inthe cytoplasmic tail of APP, Gal4.VP16 can only act as a transcriptionfactor if APP is cleaved by γ-secretase, and if the resulting productenters the nucleus. Gal4.VP16 was inserted into full-length APP695 atthe cytoplasmic boundary of the TMR, the resulting APP-Gal4.VP16 fusionprotein was transfected into a variety of cell lines (PC12, HEK293, COS,or HeLa cells), and transactivation of transcription from aco-transfected Gal4-dependent reporter plasmid encoding luciferase wasmeasured. Isolated Gal4.VP16 (without APP) was employed as a positivecontrol, and Gal4 alone (without VP16 and APP) as a negative control. Inall experiments, cells were co-transfected with a constitutiveβ-galactosidase expression plasmid in order to control for transfectionefficiency, and verified protein expression by immunoblotting.Transfections and analyses were performed as described in Example 1.

[0163] Full-length APP-Gal4.VP16 (APP-GV) transactivated Gal4-dependenttranscription much stronger than Gal4 alone in all cell types tested(˜500-2,000 fold activation depending on cell type). Surprisingly,full-length APP-Gal4.VP16 was as powerful in activating Gal4-dependenttranscription as free Gal4.VP16 (GV) without APP (FIG. 1A #1-3).Immunoblotting revealed that the transfected proteins were expressedwell, and that APP-Gal4.VP16 was partly cleaved by α- or β- andγ-secretases in the cells, resulting in stable C-terminal fragmentswhich could be detected by antibodies to Gal4 and to the cytoplasmictail of APP. A chimeric protein in which Gal4.VP16 was only fused to thecytoplasmic tail of APP without the TMR and extracellular sequences ofAPP (APPct-GV) was more potent in transactivation than full-lengthAPP-Gal4.VP16, or even Gal4.VP16 alone (˜3,000 vs. ˜1,000 foldactivation; FIG. 1A #5). In contrast, the cytoplasmic APP tailcontaining only Gal4 without VP16 (APPct-Gal4) was only slightly moreactive than Gal4 alone (<5 fold activation; FIG. 1A #8). Together theseresults show that the cytoplasmic tail of APP released by γ-cleavage iscompetent to enter the nucleus, and may partly activate transcription.Similar results were obtained in all cells tested, and thus are not aunique property of a particular cell line.

[0164] To rule out the possibility that transactivation by APP-Gal4.VP16in transfected cells may be caused by non-specific proteolysis of theAPP-Gal4.VP16 fusion protein instead of γ-cleavage, the γ-cleavageproduct of APP with an inserted Gal4.VP16 module in the cytoplasmic tail(APP γ-GV) was directly expressed, and its ability to activateGal4-dependent transcription was measured (FIG. 1A #6). Similar to thecytoplasmic tail fragment of APP, the isolated γ-cleavage product wasmore active in transcription than full-length APP-Gal4.VP16 or Gal4.VP16alone, confirming that the hydrophobic residues in the γ-cleavageproduct do not inhibit transactivation.

[0165] EXAMPLE 3

[0166] Sequence-specificity of transactivation mediated byAPP-Gal4.VP16. The cytoplasmic tail of APP contains a conserved NPTYsequence that constitutes a binding site for the PTB-domains of at leastthree proteins, Fe65, Mints/X11s, and Disabled (Fiore et al. (1995) J.Biol. Chem. 270, 30853-30856; McLoughlin et al. FEBS Lett. 397, 197-200;Borg et al. (1996) Mol. Cell. Biol. 16, 6229-6241). Binding of theseproteins to APP could contribute to the transcriptional activationmediated by APP-Gal4.VP16 by influencing γ-cleavage of APP, or byparticipating in nuclear translocation. This possibility was examined bymutating the NPTY sequence in the cytoplasmic tail of APP to NATA.Transactivation assays showed that in all cell types tested, theNPTY-mutants of Gal4.VP16 fusion proteins were as potent as wild typeproteins in activating transcription (FIG. 1A #4, 7 & 9), suggestingthat the NPTY motif and its binding proteins are not essential fortransactivation by the Gal4.VP16 module inserted into APP. In agreementwith this conclusion, co-transfection of Fe65 did not cause a majorchange in transactivation by APP-Gal4.VP16.

[0167] The specificity of transactivation by Gal4.VP16 inserted into amembrane protein was examined by introducing Gal4.VP16 into thecytoplasmic tail of neurexin 1β as a control protein that is alsoexpressed on the neuronal cell-surface but is not known to be processedby proteolytic cleavage (Ushkaryov et al. (1992) Science 257, 50-56).Neurexin 1β-Gal4.VP16 (NRX-GV) did not exhibit transactivation incontrast to APP-Gal4.VP16 (FIG. 1B, #3 & 10), suggesting that not allcell-surface Gal4.VP16-fusion proteins are competent fortransactivation. In these experiments, a dominant negative mutant ofpresenilin 2 (Steiner et al. (1999) J. Biol. Chem. 274, 28669-28673) wasco-transfected with the Gal4.VP16-fusion proteins to test if presenilinsare involved. As expected, transactivation by full-length APP-Gal4.VP16was inhibited by the presenilin 2 mutant, whereas the small amount ofresidual transactivation observed with neurexin 1β-Gal4.VP16 wasinsensitive to presenilin 2 (FIG. 1B, #3 & 10).

[0168] To determine which APP sequences enable the inserted Gal4.VP16 toactivate transcription, chimeric proteins containing differentcombinations of the extracellular and intracellular sequences of APP andof neurexin 1β were produced. When extracellular sequences and TMR ofAPP were fused to Gal4.VP16 and to the cytoplasmic tail of neurexin 1β(APPe-GV-NRXc), potent transactivation was observed. In contrast, thereverse fusion protein of the extracellular domain and TMR of neurexin1β with the cytoplasmic tail of APP (NRXe-GV-APPc) was inactive (FIG.1B, #11 and 12). Again, presenilin 2 inhibited the chimeric proteincontaining the extracellular domain of APP but had no effect on theresidual transactivation observed with the protein containing theextracellular domain of neurexin 1β, supporting the notion that specificAPP-sequences are required for transactivation as studied by this assay.

EXAMPLE 4

[0169] Fe65 binding to the cytoplasmic tail of APP stimulatestranscription. To identify co-factors that may be involved in nuclearsignaling with the cytoplasmic tail of APP, yeast two-hybrid screens forproteins that bind to the cytoplasmic tail of APP were performed asdescribed in Example 1. Similar to previous screens (26-31), Fe65 wasthe major interacting protein identified, although it was isolated at anunexpectedly high frequency (90% of all clones).

[0170] A further assay was performed to determine whether Fe65represents a co-factor for APP in nuclear signaling. Without Fe65,APP-Gal4 activated Gal4-dependent transcription only weakly (<10 fold).By contrast, co-expression of Fe65 with APP-Gal4 powerfully stimulatedtranscription (200-2,000 fold depending on the cell type). This wasobserved in all cell lines tested (PC12, HEK293, COS, or HeLa cells)(FIGS. 2A-2C). As a control, co-expression of mint 1/X11 which alsobinds to the cytoplasmic tail of APP (See, e.g., Fiore et al. (1995) J.Biol. Chem. 270; 30853-30856), had no major effect on transactivationunder these conditions. Neither Fe65 nor mint1/X11 changed thetranscription of the control β-galactosidase plasmid co-transfected intoall cells.

[0171] To examine whether Fe65 still stimulates transactivation when theFe65-binding site in the cytoplasmic tail of APP (the NPTY sequence) ismutated, yeast two hybrid assays and co-immunoprecipitations wereperformed as described in Example 1. Replacing the NPTY sequence withNATA abolished Fe65 binding as shown by yeast two-hybrid assays andco-immunoprecipitations (see also FIG. 5 below). In agreement with adirect role for Fe65 binding in stimulating transactivation by thecytoplasmic tail of APP, the same mutation also abolished theFe65-dependent stimulation of transcription (FIG. 2A).

[0172] The hypothesis that Fe65 binds to the cytoplasmic γ-cleavageproduct of APP to activate Gal4-dependent transcription in the nucleuswas confirmed by the observation that Fe65 powerfully stimulatedtransactivation by a “precleaved” APP-Gal4 fragment corresponding to theγ-cleavage product (200-2,000 fold stimulation of transactivationdepending on cell type; FIG. 2B). The effect of Fe65 depended on theintact Fe65-binding site in the cytoplasmic tail of APP (FIG. 2B). Therelative activity of APP-Gal4 co-transfected with Fe65 compares well tothat of the potent Gal4.VP16 fusion protein, suggesting that Fe65 is apowerful transcriptional activator. Further evidence for the notion thatFe65 needs to bind to the cytoplasmic APP tail in order to stimulatetranscription was obtained with series of chimeric APP/neurexinconstructs (FIG. 2C). When the cytoplasmic tail of APP-Gal4 was replacedwith the cytoplasmic tail of neurexin 1β. Fe65 did not stimulatetransactivation. However, when the 32 amino acid Fe65-binding site fromthe cytoplasmic tail of APP was transplanted into the middle of theneurexin cytoplasmic tail, powerful stimulation of transcription (>200fold over Gal4) was observed (FIG. 2C).

[0173] To ensure that APP-Gal4 is indeed cleaved at the 7-secretase sitein the transfected cells, the size of the APP-Gal4 cleavage products wasexamined. COS cells were transfected with Gal4-fusion proteins offull-length APP (as test protein), and of the cytoplasmic tail and theγ-cleavage product of APP (as size standards to identify the correctcleavage product) as described above. Immunoblotting of the transfectedcells with antibodies to the cytoplasmic tail of APP and to Gal4detected two major APP cleavage products (FIG. 3). A fragment that wasbigger than the γ-cleavage product, tentatively identified as the α- orβ-secretase product, and a fragment that of precisely the same size asthe γ-cleavage product but slightly larger than the cytoplasmic tailprotein alone (FIG. 3) were detected. No artifactual cleavage at theboundary of the TMR and the inserted Gal4 protein was detected, whilethe γ-secretase cleavage product was abundantly produced. Furthermore,analyses of cells co-transfected with Fe65 revealed that Fe65 had noapparent effect on the production of the α- and γ-cleavage products.

EXAMPLE 5

[0174] Fe65 stimulates transactivation independent of the DNA bindingprotein. Gal4 contains an intrinsic nuclear localization signal (Silveret al. (1984) Proc. Natl. Acad. Sci. USA 81, 5951-5955) andtheoretically could cause non-specific transcriptional activation thatcould be unrelated to the normal functions of these proteins. To excludeGal4-specific artifacts, APP was fused to the bacterial LexA DNA-bindingprotein (Smith et al. (1988) EMBO J. 7, 3975-3982), and measuredFe65-dependent transactivation with a LexA-dependent luciferase reporteras described in Example 3 (FIG. 4). In the absence of Fe65, APP-LexA didnot mediate significant transactivation (FIG. 4B). However,co-transfection of Fe65 strongly stimulated transactivation (40-100fold). The effect of Fe65 was less pronounced with LexA than with Gal4,possibly because the bacterial LexA DNA-binding domain acting on abacterial promoter sequence is less optimal for mammalian transcription.Similar to the Gal4 system, however, Fe65 stimulated transactivationboth with full-length APP and with the isolated cytoplasmic tail, andstimulation depended on the intact Fe65-binding site in the cytoplasmictail of APP.

EXAMPLE 6

[0175] The WW-domain and both PTB-domains of Fe65 are required fortransactivation. Fe65 is a multidomain protein that contains anegatively charged N-terminal sequence with no homology to otherproteins, a central WW-domain, and two C-terminal PTB-domains (Ermekovaet al. (1998) Adv. Exp. Med. Biol. 446, 161-180; McLoughlin et al.(1998) Biochem. Soc. Trans. 26, 497-500). The WW-domain of Fe65interacts with the cytoskeletal adaptor protein mena (Ermekova et al.(1997) J. Biol. Chem. 272, 32869-32877). The second PTB-domain of Fe65(PTB2) binds to the cytoplasmic tails of APP and other cell-surfaceproteins containing NPxY motifs (Fiore et al. (1995) J. Biol. Chem. 270:30853-30856). In addition to these cytoplasmic activities, Fe65 has beenimplicated in nuclear functions. Fe65 is partly localized to thenucleus, its first PTB-domain (PTB1) binds to the transcription factorCP2/LSF/LBP1, and its negatively charged N-terminal sequences stimulatesGal4-dependent transcription (Duilio et al. (1991) Nucleic Acids Res.19, 5269-5274). The foregoing data establish that the γ-cleavage productof APP forms a complex with Fe65 that transactivates a heterologouspromoter, suggesting that the APP/Fe65 complex functions as atranscriptional activator.

[0176] To investigate how the APP/Fe65 complex activates transcription,a series of Fe65 deletion mutants were constructed and their ability tostimulate transactivation was determined as described in Example 1. BothAPP-Gal4 and APP-LexA were used to ensure that the effects observed werenot peculiar to a particular DNA-binding protein (FIG. 5). The WW-domainand both PTB-domains of Fe65 were found to be essential for activatingtranscription. By contrast, deletion of the N-terminal third of Fe65with the acidic region suspected of activating transcription had noeffect on transactivation (FIGS. 5A and 5B). The results indicate thatin addition to the binding of the second PTB domain of Fe65 to thecytoplasmic tail of APP, the WW-domain and the first PTB domain alsointeract with target molecules in order for Fe65 to stimulatetranscription.

[0177] The Fe65 deletion mutants suggest that Fe65 is a true adaptorprotein in transcriptional regulation. To exclude possible artifactsinduced by the deletions, immunoblotting was performed to confirm thatall of the transfected proteins were stably expressed, and notprematurely degraded. Immunoprecipitations from COS cells whichco-express wild type or mutant Fe65 and APP-Gal4 showed that deletion ofthe first PTB-domain in Fe65 does not impair its ability to bind to thecytoplasmic tail of APP as long as that tail contains a wild type NPTYsequence (FIGS. 5C and 5D). Finally, point mutations in the WW-domainwere used instead of a large deletion to assess the need for the WWdomain in the stimulation of transactivation. Substitution of one of theconserved tryptophan residues of the WW-domain had no effect, whilereplacement of the central YYW motif with alanine residues abolished theFe65-dependent stimulation of transcription (FIGS. 5A and 5B). Again,immunoblotting confirmed that all mutants were stably expressed, andnone of the Fe65 proteins influenced basal transcription from theco-transfected control plasmids. Together these experiments show thatall three canonical domains of Fe65 are required to activatetranscription in a complex with the cytoplasmic tail of APP,establishing that Fe65 is a genuine adaptor protein which links multiplecomponents into a single active complex.

EXAMPLE 7

[0178] Fe65 binds to the histone acetyl transferase Tip60. As an adaptorprotein, Fe65 presumably directly or indirectly interacts withtranscription factors when it activates transcription. A candidate forsuch a binding protein is the transcription factor LBP/CP2/LSF whichinteracts with the first PTB-domain of Fe65 (Duilio et al. (1991)Nucleic Acids Res. 19, 5269-5274). However, only a weak interactionbetween LBP/CP2/LSF and Fe65 was observed in quantitative yeasttwo-hybrid assays, and no change in the amount of transactivation wasdetected when LBP/CP2/LSF was co-transfected with Fe65 and APP-Gal4.Other Fe65-interacting proteins were searched for using yeast two-hybridscreens as described in Example 1. A single prey clone that stronglybound to the first PTB-domain of Fe65, and that contains almost theentire coding sequence for Tip60, was identified. Tip60 is a histoneacetyl transferase that is expressed in two alternatively spliced forms(Tip60α and β), interacts with multiple transcription factors, and ispart of a large complex in the nucleus (Kamine et al. (1996) Virology216, 357-366). Quantitative yeast two-hybrid assays, GST-pulldownstudies, and co-immunoprecipitation experiments confirmed a stronginteraction of Fe65 with both the partial rat Tip60β obtained in theyeast two-hybrid screens, and with full-length human Tip60β (FIGS. 6Aand 6B). Furthermore, immunofluorescence analyses of transfected cellsshowed that Fe65 and Tip60β colocalize in the nucleus in a speckledpattern, suggesting that they function as a complex (FIGS. 6C-6F).

[0179] PTB-domains usually bind to NPxY target sequences, althoughvariant binding sequences have also been observed (Zwahlen et al. (2000)EMBO J. 19, 15005-15015). In a search for a possible PTB-domain targetsequence in Tip60, a single motif was detected that is remotely similarto the NPxY sequence (NKSY; residues 257-260). Mapping of the NKSYsequence onto the three-dimensional structure of Esa1, a yeast histoneacetyl-transferase whose three-dimensional structure has been solved(Yan et al. (2000) Mol. Cell 6, 1195-1205), suggests that the NKSY motifin Tip60 is located on a surface loop of a conserved domain, and thusaccessible for a binding partner. To test if the Fe65 PTB 1-domain bindsto this site, the Tip60 NKSY sequence was mutated into NASA, and theability of mutant Tip60 to bind to Fe65 was tested. No binding wasobserved for the mutant as measured either by quantitative yeasttwo-hybrid assays or GST-pulldowns (FIGS. 6A and 6B), suggesting thatthe first PTB-domain of Fe65 binds to the NKSY sequence in Tip60.

EXAMPLE 8

[0180] Binding of the APP/Fe65 complex to Tip60 mediatestransactivation. To test if Fe65 enhances transactivation by binding tothe Tip60 complex, a Gal4-Tip60 fusion protein was constructed, and theeffects of Fe65 and APP on Gal4-dependent transactivation mediated bythe Gal4-Tip60 fusion protein were examined (FIG. 7). Gal4-Tip60 alonewas unable to support significant Gal4-dependent transcription (noactivation over Gal4 alone). Co-expression of either Fe65 or APPindividually with Gal4-Tip60 did not enhance transactivation. However,when Gal4-Tip60 was co-expressed with both Fe65 and APP, transactivationwas stimulated dramatically (˜100 fold; FIG. 7). Mutant APP that isunable to bind to Fe65 (APP*) was largely inactive (˜10 fold enhancementof transactivation). Furthermore, no potentiation of transactivation wasobserved when Fe65 and APP were co-expressed with mutant Gal4-Tip60(Gal4-Tip60*) that is unable to bind to Fe65, or with Gal4 only.Together these data show that the cytoplasmic tail of APP has a directactive role in stimulating transactivation, and that it collaborateswith Fe65 in enhancing transcription by Gal4-Tip60.

[0181] In experiments described above (FIG. 5), it was found that allthree canonical Fe65 domains (the WW domain and the two PTB domains) arerequired for Fe65 to potentiate transactivation by Gal4- and LexA-APPproteins. In order to test if the same applies for the Fe65- andAPP-dependent transactivation by Gal4-Tip60, a series of Fe65 mutantswas examined in this assay (FIG. 7). The N-terminal sequence of Fe65 wasnot needed for potentiating Gal4-Tip60 dependent transactivation,whereas the second PTB-domain that binds to APP was essential. The WWdomain of Fe65 was also found to be indispensable.

[0182] The foregoing data provide a model for the function of APP andits homologs whereby proteolytic cleavage of APP releases thecytoplasmic tail to activate a nuclear signal in transcription (FIG. 8).

[0183] All references cited herein are incorporated herein in theirentirety.

1 16 1 13 PRT artificial sequence synthetic peptide 1 Gln Leu Thr ValSer Pro Glu Phe Ala Pro Pro Thr Asp 1 5 10 2 15 PRT artificial sequencesynthetic peptide 2 Asp Glu Tyr Gly Gly Gly Ile Pro Pro Gly Gln Tyr ThrSer Ile 1 5 10 15 3 16 PRT artificial sequence synthetic peptide 3 MetLeu Lys Lys Lys Pro Leu Ala Ser Ser Arg Met Lys Leu Leu Ser 1 5 10 15 419 PRT artificial sequence synthetic peptide 4 Gln Leu Thr Val Ser ProGlu Phe Pro Gly Ile Pro Pro Gly Gln Tyr 1 5 10 15 Thr Ser Ile 5 16 PRTartificial sequence synthetic peptide 5 Met Tyr Lys Tyr Arg Thr Leu AlaSer Ser Arg Met Lys Leu Leu Ser 1 5 10 15 6 15 PRT artificial sequencesynthetic peptide 6 Asp Glu Tyr Gly Gly Gly Ile Pro Pro Gly Tyr Lys TyrArg Asn 1 5 10 15 7 19 PRT artificial sequence synthetic peptide 7 GlnLeu Thr Val Ser Pro Glu Phe Pro Gly Ile Pro Pro Gly Tyr Lys 1 5 10 15Tyr Arg Asn 8 12 PRT artificial sequence synthetic peptide 8 Glu Gly SerTyr His Ile Asp Asp Ala Ala Val Thr 1 5 10 9 11 PRT artificial sequencesynthetic peptide 9 Glu Gln Met Gln Asn Ile Asp Glu Ser Arg Asn 1 5 1010 18 PRT artificial sequence synthetic peptide 10 Asn Gly Asp Trp LeuGlu Phe Pro Gly Ile Pro Pro Gly Gln Tyr Thr 1 5 10 15 Ser Ile 11 14 PRTartificial sequence synthetic peptide 11 Met Leu Lys Lys Lys Pro Leu AlaLys Met Lys Ala Leu Thr 1 5 10 12 4 PRT artificial sequence syntheticpeptide 12 Asn Lys Ser Tyr 1 13 4 PRT artificial sequence syntheticpeptide 13 Asn Ala Thr Ala 1 14 4 PRT artificial sequence syntheticpeptide 14 Asn Ala Ser Ala 1 15 4 PRT artificial sequence syntheticpeptide 15 Asn Lys Ser Tyr 1 16 37 PRT artificial sequence syntheticpeptide 16 Ser Asp Leu Pro Ala Gly Trp Met Arg Val Gln Asp Thr Ser GlyThr 1 5 10 15 Tyr Tyr Trp His Ile Pro Thr Gly Thr Thr Gln Trp Glu ProPro Gly 20 25 30 Arg Ala Ser Pro Ser 35

We claim:
 1. A method of identifying an agent that affects the cleavageof amyloid-β precursor protein (APP) comprising providing a cellcontaining APP modified in the C-terminal cytoplasmic tail to allowdetection of nuclear localization of said cytoplasmic tail; contactingsaid cell with a candidate agent; and measuring nuclear localization ofsaid C-terminal cytoplasmic tail, wherein an increase or decrease innuclear localization in the presence of said candidate agent relative tonuclear localization in the absence of said candidate agent isindicative of an agent that affects the cleavage of APP.
 2. The methodof claim 1 wherein APP is modified to comprise a DNA-binding domain of atranscription factor and a transcriptional activator of the same or adifferent transcription factor, and wherein said cell further containsan indicator gene operably linked to a nucleic acid comprising a bindingsite for said DNA-binding domain.
 3. The method of claim 2 wherein saidDNA-binding domain is Gal4 or LexA.
 4. The method of claim 2 whereinsaid transcriptional activator is VP16.
 5. The method of claim 1 whereinsaid APP is modified to comprise a DNA-binding domain of a transcriptionfactor and wherein said cell further comprises an indicator geneoperably linked to a nucleic acid comprising a binding site for saidDNA-binding domain, and wherein Fe65 is provided to said cell.
 6. Themethod of claim 5 wherein said DNA-binding domain is Gal4 or LexA. 7.The method of claim 2 wherein nuclear localization is measured bymeasuring expression of said indicator gene.
 8. The method of claim 5wherein nuclear localization is measured by measuring expression of saidindicator gene.
 9. The method of claim 1 wherein said cell is aeukaryotic cell.
 10. The method of claim 1 wherein said cell is amammalian cell.
 11. The method of claim 1 wherein said cell is a humancell.
 12. A method of identifying an agent that affects the cleavage ofamyloid-β precursor protein (APP) comprising co-transfecting a cell with(a) a nucleic acid encoding a modified APP, wherein said modified APPcomprises Gal4 and VP16 in the C-terminal cytoplasmic tail, and (b) anucleic acid encoding an indicator gene under the control of one or morecopies of a Gal4 regulatory element; contacting said cell with acandidate agent; and measuring expression of said indicator gene,wherein an increase or decrease in expression in the presence of saidcandidate agent relative to the absence of said agent is indicative ofan agent that affects cleavage of APP.
 13. A method of identifying anagent that affects the cleavage of amyloid-β precursor protein (APP)comprising providing a cell containing APP and Tip60 modified to allowdetection of nuclear localization of a C-terminal cytoplasmic cleavageproduct of APP; contacting said cell with a candidate agent; andmeasuring nuclear localization of said C-terminal cytoplasmic cleavageproduct, wherein an increase or decrease in nuclear localization in thepresence of said agent relative to nuclear localization in the absenceof said agent is indicative of an agent that affects the cleavage ofAPP.
 14. The method of claim 13 wherein said Tip60 is modified by fusionwith the DNA binding domain of a transcriptional activator, and whereinsaid cell further contains Fe65.
 15. The method of claim 14 wherein saidDNA binding domain is Gal4 or LexA.
 16. The method of claim 14 whereinsaid cell further contains an indicator gene operably linked to anucleic acid comprising a binding site for said DNA binding domain. 17.The method of claim 16 wherein said nuclear localization is measured bymeasuring expression of said indicator gene.
 18. The method of claim 13wherein said cell is a eukaryotic cell.
 19. The method of claim 13wherein said cell is a mammalian cell.
 20. The method of claim 13wherein said cell is a human cell.
 21. A method of identifying an agentthat affects the cleavage of amyloid-β precursor protein (APP)comprising co-transfecting a cell with (a) a nucleic acid encoding APP,(b) a nucleic acid encoding Fe65, (c) a nucleic acid encoding a fusionprotein of Tip60 and Gal4, and (d) a nucleic acid encoding an indicatorgene under the control of one or more copies of a Gal4 regulatoryelement; contacting said cell with a candidate agent; and measuringexpression of said indicator gene, wherein an increase or decrease inexpression in the presence of said candidate agent relative to theabsence of said agent is indicative of an agent that affects thecleavage of APP.
 22. A vector comprising a first nucleic acid encodingamyloid-β precursor protein (APP) operably linked to a promoter, whereina second nucleic acid encoding a heterologous DNA-binding domain of atranscription factor is contained within the region of the first nucleicacid that encodes the C-terminal cytoplasmic tail of APP.
 23. Avector-comprising a nucleic acid encoding amyloid-β precursor protein(APP) operably linked to a promoter wherein a nucleic acid moduleencoding a heterologous DNA-binding domain of a transcription factor anda transcriptional activator of the same or a different transcriptionfactor is contained within the region of the nucleic acid that encodesthe C-terminal cytoplasmic tail of APP.
 24. The vector of claim 28wherein the DNA-binding domain is Gal4 or LexA.
 25. The vector of claim23 wherein the DNA-binding domain is Gal4 or LexA and thetranscriptional activator is VP16.
 26. A vector comprising a nucleicacid encoding Tip60 and a heterologous DNA binding domain of atranscription factor.
 27. The vector of claim 26 wherein said DNAbinding domain is Gal4.
 28. A cell comprising the vector of claim 22.29. A cell comprising the vector of claim
 23. 30. A cell comprising thevector of claim
 24. 31. An agent that affects the cleavage of amyloid-βprecursor protein (APP) identified by a method comprising providing acell containing APP modified in the C-terminal cytoplasmic tail to allowdetection of nuclear localization of said cytoplasmic tail; contactingsaid cell with a candidate agent; and measuring nuclear localization ofsaid C-terminal cytoplasmic tail, wherein an increase or decrease innuclear localization in the presence of said candidate agent relative tonuclear localization in the absence of said candidate agent isindicative of an agent that affects the cleavage of APP.
 32. An agentthat affects the cleavage of amyloid-β precursor protein (APP)identified by a method comprising providing a cell containing APP and aprotein that interacts with APP to activate transcription, wherein theprotein is modified to allow detection of nuclear localization of aC-terminal cytoplasmic cleavage product of APP; contacting said cellwith a candidate agent; and measuring nuclear localization of saidC-terminal cytoplasmic cleavage product, wherein an increase or decreasein nuclear localization in the presence of said agent relative tonuclear localization in the absence of said agent is indicative of anagent that affects the cleavage of APP.
 33. A composition comprising theagent of claim
 31. 34. A composition comprising the agent of claim 32.35. A kit comprising a first compartment containing cells comprising avector wherein said vector comprises a nucleic acid encoding amyloid-βprecursor protein (APP) operably linked to a promoter wherein a nucleicacid module encoding a heterologous DNA-binding domain of atranscription factor and a transcriptional activator of the same or adifferent transcription factor is contained within the region of thenucleic acid that encodes the C-terminal cytoplasmic tail of APP. 36.The kit of claim 35 wherein said cells further contain a nucleic acidencoding an indicator gene under the control of a regulatory element forsaid DNA-binding domain.
 37. A kit comprising a first compartmentcontaining a vector comprising a nucleic acid encoding amyloid-βprecursor protein (APP) operably linked to a promoter wherein a nucleicacid module encoding a heterologous DNA-binding domain of atranscription factor and a transcriptional activator of the same or adifferent transcription factor is contained within the region of thenucleic acid that encodes the C-terminal cytoplasmic tail of APP. 38.The kit of claim 37 further comprising a second compartment containing areporter plasmid comprising a nucleic acid encoding an indicator geneunder the control of a regulatory element for said DNA-binding domain.